We previously performed a genome-wide analysis designed to identify linkage of susceptibility to subacute (0.3 ppm, 48-72 hr) O3-induced inflammation (polymorphonuclear leukocyte PMN influx) to any chromosomal intervals that may contain within them genes that are polymorphic between O3-susceptible C57BL/6J (B6) and O3-resistant C3H/HeJ (C3) mice. We found a significant QTL on chromosome 17 (Inf2) and a suggestive locus on chromosome 11. Within Inf2 are the candidate genes Tnf (tumor necrosis factor-a, Tnf), Lta (lymphotoxin-a), and H2 (histocompatibility loci). Because Tnf is a proinflammatory cytokine postulated to have a role in oxidant-related lung injury, we investigated and confirmed Tnf as a candidate gene for determination of differential O3-induced inflammation in B6 and C3 mice. A number of potential candidate genes are also located within the chromosome 11 QTL, however given the strength of linkage association with Inf2, we have focused our efforts initially on this QTL. [unreadable] [unreadable] To further investigate the mechanisms through which TNFR modulates O3-induced lung injury. One investigation was designed to determine the molecular mechanisms of TNF receptor (TNF-R)-mediated cell signaling and lung injury induced by O3. We found that O3 significantly activated lung NF-kappaB in Tnfr wt mice before the development of lung injury. Basal and O3-induced NF-kappaB activity was suppressed in Tnfr(-/-) mice. Compared with Tnfr(+/+) mice, MAPKs and activator protein (AP)-1 were lower in Tnfr(-/-) mice basally and after O3. Furthermore, inflammatory cytokines, including macrophage inflammatory protein-2, were differentially expressed in Tnfr(-/-) and Tnfr wt mice after O3. O3-induced lung injury was significantly reduced in Nfkb1(-/-) and Jnk1(-/-) mice relative to respective control animals. Our results suggest that NF-kappaB and MAPK/AP-1 signaling pathways are essential in TNF-R-mediated pulmonary toxicity induced by O3.[unreadable] [unreadable] We have also initiated studies to reduce the size of Inf2 to determine whether additional candidate genes emerged. To reduce this region and search for candidate genes, O3-induced inflammation in congenic 2R (H2d), 4R (H2b), and 5R (H2d) mice were compared to those of similarly exposed O3-susceptible background B10 and O3resistant donor A strains. The congenic regions on the 2R, 4R, and 5R mice overlap (tile) varying congenic regions within Inf2 and thus enable subtractive discrimination of the locus. Comparison of the inflammatory responses of the congenic strains with respective congenic regions indicated that the 18.6 19.0 cM region of chromosome 17 accounted for a major portion of the inflammatory response to O3. Because of the prevalence of MHC class II genes in Inf2, we asked whether they are candidate susceptibility genes for O3-induced inflammation. O3-induced inflammation in MHC II -/- mice deficient in MHC class II genes (H2-Abl, -Aa, -Eb1, -Eb2, and -Ea) were compared to that in MHC II +/+ mice. Significantly greater O3-induced inflammation was found in MHC II +/+ mice compared to MHC II -/- mice. These are the first studies to conclusively demonstrate a role for MHC class II genes in oxidant-induced lung inflammation. Interestingly, these studies excluded Tnf from the reduced Inf2. The novel role of MHC class II genes in susceptibility to O3 provides unique insight to the mechanisms of O3 effects in the lung. [unreadable] [unreadable] We have also used a candidate gene approach to investigate the role of other mediators involved in the process of O3-induced inflammation to better understand differential susceptibility. Matrix metalloproteinases (MMPs) havee an important role in many physiological and pathological conditions, including acute lung injury, asthma, and chronic obstructive pulmonary diseases. We hypothesized that MMPs are important in the pathogenesis of O3-induced airway inflammation and compared the lung injury responses in either Mmp7-deficient (Mmp7-/-) or Mmp9-deficient (Mmp9-/-) mice and their wild-type (+/+) controls. Relative to air-exposed controls, MMP-9 activity in BAL fluid was significantly increased by O3 in Mmp9+/+ mice. O3-induced inflammation was significantly greater in Mmp9-/- mice, compared with Mmp9+/+ mice. KC and MIP-2 levels in BALF were also significantly higher in Mmp9-/- mice than in Mmp9+/+ mice after O3, though no differences in mRNA expression for these chemokines were found between genotypes. Parameters of inflammation were not significantly different between Mmp7-/- and Mmp7+/+ mice after O3. Results thus demonstrated a protective role of MMP-9, but not MMP-7, in O3-induced lung inflammation and injury. The mechanism through which Mmp9 limits O3-induced airway injury is not known, but may be via post-transcriptional effects on proinflammatory CXC chemokines including KC and MIP-2. [unreadable] [unreadable] In collaboration with Les Kobzik at Harvard, we have also begun to investigate the mechanisms through which TLR4 modulates O3-induced inflammation. We used microarray analyses to find that macrophage receptor with collagenous structure (MARCO) was upregulated after O3 exposure in mice with a mutation in TLR4 relative to mice with normal TLR4. We then found that after ozone exposure, MARCO-/- mice showed greater lung injury than did MARCO+/+ mice. Further, intratracheal instillation of oxidized lipids known to be created by ozone exposure caused substantial neutrophil influx in MARCO-/- mice, but had no effect in MARCO+/+ mice. These results led us to conclude that MARCO has an important role in decreasing pulmonary inflammation after oxidant inhalation by scavenging proinflammatory oxidized lipids from lung lining fluids.[unreadable] [unreadable] Interleukin 10 (IL-10) is a pleiotropic anti-inflammatory cytokine produced by activated monocytes and macrophages, helper T cells, and B cells. Increased IL-10 production downregulates TNF-a&#61472;and IL-6, and inhibits MIP-2. IL-10 also inhibits iNOS production in macrophages, and blocks NF-kB&#61472;activation. We tested the hypothesis that murine IL-10 protects lungs from O3-induced inflammation. Il10-deficient (Il10-/-) and wild type (Il10+/+) mice were exposed to 0.3-ppm O3 or filtered air and O3-induced inflammation was significantly greater in Il10-/- mice than in Il10+/+ mice. O3-enhanced production of MIP-2 and CD86 and NF-kB activity was greater in the lungs of Il10-/- compared with Il10+/+ mice. Microarray analysis of lungs from O3-exposed Il10-/- and Il10+/+ mice identified effector genes of Il10, including IL17r, Lmod2, and Scg5. Thus, IL-10 protects the lung against O3-induced inflammation and potential mechanisms through which this effect is mediated have provided additional novel insight to the role of this gene in susceptibility to O3-induced inflammation.[unreadable] [unreadable] In collaboration with Dr. Dave Peden (UNC), nine healthy nonsmoking volunteers (21-30 yr old) completed a double blind crossover study in which they were exposed to either filtered air (FA) or 0.4 ppm O3 for 2 hr (with intermittent exercise) during 2 study sessions (separated by at least 2 wk). We found that O3 increased the number of airway monocytes and significant upregulation of cell surface molecules associated with innate immunity and antigen presentation on airway monocytic cells. These data are consistent with our studies in mice that MHC Class II genes are important determinants of susceptibility to O3-induced inflammation. The data also suggest that O3 causes innate immune priming of airway monocytes, increased capacity to present processed antigens to CD4+ T T cells, and an overall increase in the population of APCs in the airways.